The influence of 57 chemicals (mineral and
organic acids, organic solvents, phenolic compounds, mineral and organic salts)
on the efficiency of protein determination by the Lowry method was assessed.
The study revealed the Lowry method to be unreliable in an acidic and
increasing vitamin C-rich medium. For organic solvents, it is advisable to
evaporate these compounds when they are used to extract proteins, prior to
proteins measurement the Lowry method. The influence of phenolic compounds on
the Lowry method was found to be governed by the structure of their molecules
(presence of double bounds, number of OH groups, presence of methyl groups, etc.),
while ammonium sulfate, which is a major agent used in the enzyme purification
process, was found to result in an overestimation of the protein content.
Therefore, its use for enzyme purification should be done with caution.

Several methods are used to
measure the protein content of different food products, enzymatic solutions and
other biological mixtures. These methods include that of Kjeldahl1,
which allows the extrapolation of protein content from the estimation of the
amount of nitrogen. In this method, the food product is mineralized with a
boiling sulfuric acid solution and the amount of ammonium ions formed is determined.
The method of Biuret2 allows the determination of protein content
from the quantification of peptide bonds with the aid of Cu2+ ion in
an alkaline environment containing chelating molecules such as tartrate or EDTA.
The method of Smith et al.3 allows the protein content
determination through their peptide bonds. Indeed, the bicinchoninic acid gives
shape to a stable complex with copper ions in an alkaline environment with a
peptide bond. This method is very similar to that of Biuret2. The
method of Bradford4 uses the Coomassie blue G 200 which reacts with
proteins particulary on the cationic groups and aromatic rings. The absorption
in the ultra-violet regions reveals the peptide bond which absorbs at around
214 nm and the aromatic groups (phenylalanine, tyrosine and moreover
tryptophane), which give a large band at 280 nm. The method of Lowry et al.5
combines the reaction of Biuret 2 with the action of a mercury
reagent of phenols, the reagent of Folin-Ciocalteus. It quantifies both of the
peptide bonds and the tyrosine residues.

Among the methods quoted, that of Lowry
seems to be the most used to determine the amounts of protein contained in
biological or chromatographic solutions. However, these environments are often
heterogeneous as they contain other molecules which can interact with the
proteins to be measured or with the reagent used. This could lead to errors in
the estimation of the protein content of these solutions. Therefore, in this work, we examined the effect
of a variety of chemicals (mineral and organic acids, organic solvents,
phenolic compounds, mineral and organic salts) on the accuracy of protein
measurement by the Lowry method.

The measurement of protein was
carried out on a total volume of 200 µl containing 50 µg of bovine serum
albumin and a variable amount of the chemical product, the influence of which
is to be determined on the effectiveness of the method of Lowry. A volume of 2
ml of the mixture (prepared with 100 µl of 0.5% (w/v) copper sulfate solution,
100 µl of 1% (w/v) potassium-sodium tartrate solution and 10 ml of 2% (w/v)
sodium carbonate solution) was added. The resulting mixture was shaken and
incubated in the dark for 15 min at ambient temperature. Then, 200 µl of
Folin-Ciocalteus reagent (half diluted in 0.1 N NaOH) was added. The mixture
was shaken once more and allowed to rest in the dark for 30 min to allow colour
development. Absorbance of mixtures was measured at 660 nm with a
spectrophotometer (GENESYS TM 5) against a control containing no
protein extract. These absorbances were further converted to
percentage of variation of the amount of protein against the absorbance of
mixture containing only 50 µg of bovine serum albumin.

RESULTS AND DISCUSSION

Influence of acids

Among the tested acids, only ascorbic acid
caused a very high overestimation of the amount of protein in the reaction
mixture (Figure 1b). For an amount of 50 µg of protein present in the reaction
mixture, we obtained an estimation of 2000 %. This overestimation increased
with the concentration of vitamin C. The presence of double bonds in the
structure of ascorbic acid may explain this result. These double bonds are
similar to those present in aromatic amino acids such as phenylalanine,
tyrosine, tryptophane and histidine. This high overestimation suggests that
protein estimation in fruit juices, which are rich in vitamin C may not be
reliable when the method of Lowry is used. Other quantitative methods for the
assessment of the amount of protein based on peptide bonds such as that of Smith
and Circles6 and Bradford4 could be preferable.

Boric acid resulted in a slight
overestimation of the amount of protein in the reaction mixture in a
concentration dependent manner, while trichloroacetic acid and oxalic acid
caused little or no variation in the amount of protein estimated irrespective
of their concentration in the medium (Figure 1b).

Other acids such as metaphosphoric acid,
hydrochloric acid, perchloric acid, propionic acid, sulfuric acid, acetic acid,
saccharic acid, glutamic acid and molybdic acid resulted in an underestimation
of the amount of protein in the reaction mixture (Figures
1 a,b, and c). This
underestimation increased with the concentration of acids in the medium.

Influence of organic solvents

With organic solvents, propanol-1 and
2-methyl-1-propanol induced an underestimation of the amount of protein in the
medium, while isobutanol hardly exerted any influence on the amount of protein
determined using Lowry method. Organic solvents resulting in an overestimation
include ethanol, methanol, acetone and hexane (Figure 2). However, more
accurate results were obtained when organic solvents used for protein
extraction were evaporated before protein quantification with Lowry method.

Influence of phenolic compounds

All tested phenolic compounds
exerted influence on the accuracy of protein estimation by the method of Lowry
(Figures 3 a and b). While only p-nitrophenol induced a slight
underestimation of the amount of protein, the others: orcinol, 2-naphtol,
naphto-resorcinol, resorcinol, phenol and 2,6-dichlorophenol caused a high
overestimation of the protein level, reaching up to 3000 % in some cases. This
highest overestimation was obtained with naphto-resorcinol, followed by
resorcinol, orcinol, phenol, 2-naphtol and 2,6-dichlorophenol in that order
(Figures 3 a and b). The overestimation of the amount of protein present in the
phenolic compounds may be due to the presence of double bonds and probably to
the number of OH grouping present in the structure of the molecule.

With monophenols, when the grouping of NO2
is grafted on the phenol, it loses its capability to interfere, but, when two
atoms of chloride are present on the phenol, increased overestimation compared
to that of phenol.

For polyphenols, the interference on
protein estimation varied according to the number of OH groups present. Indeed,
naphto-resorcinol containing three groups of OH in its structure showed the
greatest interference, followed by resorcinol which has two OH groups. As for
orcinol, it also possesses two OH groups but the presence of methyl group
reduced the overestimation power of the molecule. 2-naphtol showed an
overestimation power higher than that of phenol, probably because it possesses
much more double bonds than phenol, since the two compounds possess only one OH
group in their structure.

Influence of salts

Among all the tested salts (27
in all), only four induced an underestimation of the amount of protein. These
were sodium hypochlorite, ammonium molybdate, sodium chloride and ammonium
persulfate
(Figures 4a, 4b, 4c, 4d
and 4e). Six others hardly exerted any effect upon the
effectiveness of the method of Lowry. These were sodium acetate, sodium
hydrogen carbonate, ammonium nitrate, calcium carbonate, ammonium vanadate and
disodic oxalate (Figures 4a, 4b, 4c, 4d
and 4e). The salt that caused the
greatest overestimation of protein include manganese chloride, potassium
hexacyanoferate, lead acetate, sodium sulfate, potassium hydroxide, ammonium
sulfate, barium chloride, copper acetate, potassium sulfate and calcium
hypochlorite. Calcium seems to play an important role in the overestimation of
the amount of protein, since sodium hypochlorite caused an underestimation
whereas calcium hypochlorite resulted in an overestimation.

Ammonium sulfate which is the most widely
used chemical in enzyme purification7-10 processes induced an
overestimation of the amount of protein. This has a lot of implications since
this salt is used to precipitate enzymes11,12 and saturate chromatographic
gels into salt13 when performing hydrophobic interaction
chromatography. Its role in that process of fractionating is to make the medium
more hydrophobic14-18. In these two situations, the enzymatic
solutions will contain this product which could produce false results when
determining the amount of protein. This overestimation of protein will wrongly
suggest a low specific activity for the enzyme, and thus a wrong conclusion
that the enzyme in question is relatively non-stable. Hence, we suggest that
for hydrophobic interaction chromatography, sodium thiosulfate can be used for
the saturation process as it has little or no effect on Lowry method.

Conclusion

The influence of 57 chemicals
(mineral and organic acids, organic solvents, phenolic compounds, mineral and
organic salts) on the effectiveness of the method of Lowry for protein
measurement was examined. The different chemical products were observed to have
different effects on the efficiency of the method of Lowry. Some
(ascorbic acid, boric acid, ethanol, methanol, acetone, hexane, oricinol,
2-naphtol, resorcinol, naphto-resorcinol, phenol, 2,6-dichlorophenol, manganese
chloride, potassium hexacyanoferate, lead acetate, sodium sulfate, potassium
hydroxide, ammonium sulfate, barium chloride, copper acetate, potassium sulfate
and calcium hypochlorite)brought about an overestimation while
others (isobutanol, sodium acetate, sodium hydrogen carbonate, ammonium
nitrate, calcium carbonate, ammonium vanadate and disodic oxalate)produced an underestimation yet others (metaphosphoric
acid, hydrochloric acid, perchloric acid, propionic acid, sulfuric acid, acetic
acid, saccharic acid, glutamic acid, molybdic acid, propanol-1,
2-methyl-1-propanol, p-nitrophenol, sodium hypochlorite, ammonium
molybdate, sodium chloride and ammonium persulfate) do not have any effect on
the effeciency of the method of Lowry. Acidity of the medium, number OH groups
and double bonds on the chemicals are found to play some roles in their capacity
to influence protein estimation.

ACKNOWLEGEMENT

The authors are grateful to Professor
Bernard Colas of University of Nantes
(France), Unité de Recherche sur la Biocatalyse,
CNRS-UMR 6204, for technical assistance during this work.